Methods and compositions for treatment of pathological hydrophobic interactions in biological fluids

ABSTRACT

In accordance with the present invention, a method and composition is provided for treating pathological hydrophobic interactions in biological fluids in which there is acute impairment of the circulation, especially the microcirculation. More particularly, the present invention relates to compositions and methods for treating circulatory diseases comprising using certain ethylene oxide-propylene oxide condensation surface active copolymers either alone or in combination other compounds. 
     Also contemplated in the present invention is a method for preserving a suspension of platelets. The method comprises adding an effective amount of a surface active copolymer. 
     The surface active copolymer can be an ethylene oxide-propylene oxide condensation product with the following general formula: 
     
         HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 
    
      O) b  H 
     wherein a is an integer such that the hydrophobe represented by (C 3  H 6  O) has a molecular weight of approximately 950 to 4000, preferably approximately 1750 to 3500, and b is an integer such that the hydrophile portion represented by (C 2  H 4  O) constitutes approximately 50% to 95% by weight of the compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 291,925 filed on Dec. 29, 1988, now U.S. Pat. No.4,879,109, which is a continuation-in-part of U.S. patent applicationSer. No. 45,459 filed on May 7, 1987, now U.S. Pat. No. 4,801,452 issuedon Jan. 31, 1989, which is a continuation-in-part of U.S. patentapplication Ser. No. 43,888 filed on Apr. 29, 1987, now abandoned, whichis a continuation of U.S. patent application Ser. No. 863,582, nowabandoned, filed on May 15, 1986.

TECHNICAL FIELD

The present invention relates to a method and composition for treatingpathological hydrophobic interactions in which there is acute impairmentof the circulation, especially the microcirculation. More particularly,the present invention relates to compositions and methods for treatingcirculatory diseases comprising using certain ethylene oxide-propyleneoxide condensation copolymers either alone or in combination with othercompounds, including but not limited to, fibrinolytic enzymes,anticoagulants, free radical scavengers, antiinflammatory agents,antibiotics, membrane stabilizers and/or perfusion media.

BACKGROUND OF THE INVENTION

The term "pathological hydrophobic interactions" means detrimentaladhesion of components, including but not limited to, cells andmolecules in blood or other biological fluids thereby slowing orstopping the flow of blood or other biological fluid. The term"fibrinolytic enzyme" menas any enzyme that is capable of cleavingfibrin or capable of causing fibrin to be cleaved. Enzymes that arecapable of cleaving fibrin or causing fibrin to be cleaved include, butare not limited to, streptokinase, urokinase, tissue plasminogenactivator (t-PA) produced from cell cultures, tissue plasminogenactivator produced by recombinant DNA technology and plasminogenactivator produced from prourokinase. The terms "isotonic" or"isoosmotic" solution are defined as solutions having the same osmoticpressure as blood. The term "SOD" means superoxide dismutase and refersto any enzyme capable of neutralizing oxygen radicals. The terms clot,fibrin clot and thrombus are used interchangeably. The term"microcirculation" means blood circulation through blood vessels thatare about 50 microns in diameter or less. The term "soluble fibrin"means soluble high molecular weight polymers of fibrinogen and fibrin.The term "biological fluids" means blood, lymph, or other fluids foundin animals or humans. The term "platelet suspension" means a suspensionof platelets that has a higher concentration of platelets than thatfound in blood. The term "plasma extender" means any substance that canbe added to animal or human blood to maintain or increase coloid osmoticpressure. The term "cytoprotective" as used herein, means an increasedability of myocardial, endothelial and other cells to withstand ischemiaor recover from ischemia, or other noxious insults including but notlimited to burns. The term "ischemic tissue" is any tissue that isdamaged from reduced blood flow. The term "anticoagulant" is anycompound or agent that inhibits the blood coagulation process. The term"reperfusion injury" means injury to tissue or cells which occurs duringreperfusion of damaged tissue with blood. The term "damaged tissue"means tissue damaged by ischemia, burns, toxins or other noxious insult.

It is to be understood that the citation of art contained herein is inno way to be construed as an admission that said art is suitablereference against the present patent application nor should thiscitation act as a waiver of any rights to overcome said art which may beavailable to the applicant.

A number of reports have described high amounts of fibrinogen and/orsoluble fibrin in the blood of patients with thrombosis, impendingthrombosis and many other diseases. These conditions include acute orchronic infection, severe trauma, burns, sickle cell crisis, malaria,leukemia, myocardial infarction, sepsis, shock, and almost any seriousillness which produces tissue damage or surgical maneuvers. Evidenceindicates that the high concentrations of fibrinogen and/or solublefibrin may play an important role in the pathology of the conditions.Furthermore, much of the pathology that is encountered in disease may bedue to pathological hydrophobic interactions which may be at leastpartially mediated by high concentration of fibrinogen and/or solublefibrin.

What is needed is a means of decreasing the adverse effects of solublefibrin. This would involve blocking the adhesion of soluble fibrin tocells in the circulation thereby blocking the aggregation of such cellsand their adhesion or friction to vessel walls in the microvasculature.This would also decrease the risk of thrombosis.

Each year about 550,000 Americans die from heart attacks. Evenmore--close to 700,000--have heart attacks and live. While a heartattack victim may survive, part of his or her heart will almostcertainly die. The death of heart muscle, called myocardial infarction,is due to coronary artery thrombosis in 70-90% of the cases. When athrombosis, or blood clot, occludes one of the arteries of the heart, itstops the flow of blood to the surrounding muscle which deprives it ofoxygen and other nutrients. In the past, nothing could be done toreverse this process. The high technology devices in intensive careunits mostly support patients so they can live while a portion of theirheart dies.

Similar situations occur in many other tissues when the blood supply tothe tissue is affected by a thrombus or embolus. Stroke, deep veinthrombosis and pulmonary embolus are examples. Typically, the clot formsand is not treated for a relatively long period of time. Blood flowdistal to the clot is greatly diminished or is stopped completely. Thetissue that is normally fed by that vessel will be severely damagedunless blood flow is reestablished in a short period of time.

It has been found that certain enzymes are able to degrade, initiate oractivate other enzymes that can degrade fibrin deposits to open cloggedarteries. The enzymes which have been used successfully includestreptokinase, urokinase, prourokinase, tissue plasminogen activatorproduced from cell cultures and tissue plasminogen activator produced byrecombinant DNA technology. These enzymes are most successful ifadministered shortly after the occlusion of the blood vessels before theheart tissue has sustained irreversible damage. In one study of 11,806patients treated with intravenous or intracoronary artery streptokinase,an 18% improvement of survival was demonstrated. If the treatment wasbegun within one hour after the initial pain onset of the heart attack,the in-hospital mortality was reduced by 47%. (See The Lancet, Vol.8478, p. 397-401, Feb. 22, 1986). It was demonstrated that early lysisof the thrombus resulted in salvage of a portion of heart tissue whichwould have otherwise have died. In studies using angiography to assessthe patency of blood vessels, it was found that tissue plasminogenactivator could completely open the vessels of 61% of the 129 patientsversus 29% of controls who were not treated with the enzyme. (SeeVerstraete, et al., The Lancet, Vol. 8462, p. 965-969, Nov. 2, 1985).Tissue plasminogen activator requires the addition of approximately 100μl of Tween 80 per liter of solution to promote dispersion of theenzyme. (See Korninger, et al., Thrombos, Haemostas, (Stuttgart) Vol.46(2), p. 561-565 (1981)).

The natural enzymes that lyse thrombi in vessels do so by activatingfibrinolysis. Fibrin is the protein produced by polymerization offibrinogen. It forms a gel which holds the thrombus together. The fibrinmolecules which form clots gradually become cross-linked to make a morestable clot. All three enzymes, urokinase, streptokinase and tissueplasminogen activator, are effective because of their ability toactivate an enzyme, plasmin, which degrades fibrin. Thus, they havesimilar effects on fibrin but they have different toxicities. If thefibrinolytic mechanisms (i.e., plasmin) are activated in the vicinity ofa clot, the clot is lysed. If, however, they are activated systemicallythroughout the circulation, the body's capacity to stop bleeding orhemorrhage is markedly reduced. Streptokinase and urokinase tend toactive systemic fibrinolysis. Consequently, they have been mosteffective when injected directly into the affected blood vessel.

Tissue plasminogen activator or t-PA, in contrast, becomes effectiveonly when it is actually attached to fibrin. This means its activity islargely localized to the immediate area of a clot and does not producesystemic fibrinolysis. For this reason, tissue plasminogen activator isthought to produce less risk of hemorrhage than the other enzymes. Ifhigh does are used in an effort to increase the rate of clot lysis or tolyse refractory clots, then the amount of systemic fibrinolysis and riskof hemorrhage can become significant. t-PA can be injected intravenouslyinto the general circulation. It circulates harmlessly until it contactsthe fibrin in a blood clot where it becomes activated and causes thelyses of the clot. Tissue plasminogen activator is able to cause thelysis of a clot which is extensively cross-linked. This means it ispossible to lyse clots which have been present for many hours.

Remarkable as the new enzyme therapies are, they are subject to seriouscomplications and are not effective in all patients. Clots in theanterior descending branch of the left coronary artery are much morereadily lysed than those in other arteries. If the enzyme is notdelivered by the blood stream directly to the thrombus, it has noeffect. For various reasons, more blood passes by or trickles aroundthrombi in the left anterior descending coronary artery than in theother major arteries. In addition, the presence of collateralcirculation which forms in response to compromised blood flow in themajor arteries adversely affects the rate of reopening or recanalizationof the thrombosed major arteries. It is thought the presence of manycollateral vessels which allow blood to bypass the clot reduces thepressure gradient across the clot. This in turn reduces the blood flowthrough the tiny openings which may persist in the clot, impedes thedelivery of enzymes to the clot, and prevents the clot from being lysed.

Even after the clot has been lysed, the factors which led to theformation of the thrombus persist. This produces a high incidence ofre-thrombosis and further infarction in the hours and days followinglysis of the clot. Rethrombosis has been reported in between 3% and 30%of cases in which the initial treatment successfully lysed the clot.Anticoagulants are currently used to prevent the formation of newthrombi, but they tend to induce hemorrhage. There is a delicate balancebetween the amount of anticoagulation necessary to prevent re-thrombosisof the vessels and that which will produce serious hemorrhage.

A reported advantage of t-PA is its short half-life of less than 10minutes, which may allow rapid reversal of bleeding problems should theyoccur. However, the clinical value of this consideration has not yetbeen demonstrated. Moreover, the short half-life may lead to anincreased reocclusion rate following discontinuation of thrombolytictherapy, (See Williams, D. O., et al., "Intravenous recombinanttissue-type plasminogen activator in patients with acute myocardialinfarction: a report from the NHLBI Thrombolysis in MyocardialInfarction Trial.", Circulation 1986; 73:338-46). To counter thisproblem, t-PA infusions have been continued for up to 6 hours in phaseII of the TIMI (Thrombolysis in Myocardial Infarction Trial). Whetherthis will effectively reduce the incidence of reocclusion withoutincreased bleeding remains to be proven. Although active thrombolysisceases shorly after discontinuing administration of t-PA, it takesseveral hours to replace fibrinogen, so that the risk of continuedbleeding does not terminate when t-PA is stopped. (See Rich, M. W.,"tPA: Is it worth the price?", American Heart Journal, 1987, Vol.114:1259-1261.

Finally, dissolving the clot after irreversible damage has taken placehas little effect. The irreversible damage could be either to the heartmuscle or vascular bed of the tissue supplied by the blood vessel. Oncea cell is dead, the change is irreversible. However, the termirreversible damage is frequently applied to tissue in which a chain ofevents leading to cell death has been initiated, even though most cellsare not yet dead. If this chain of events were broken, for example byrestoring the microvasculature blood supply or stabilizing fragilemembranes, then many cells could be saved. A major problem in widespreadimplementation of this new enzyme therapy is to find ways of identifyingand treating the patients earlier in their disease and to find ways tomake the treatment effective for a longer period of time after theinitiation of thrombosis.

Animal studies have provided a better understanding of the events whichcontrol blood flow and tissue death following coronary arterythrombosis. Much of the heart muscle receives blood from more than onevessel. For this and other reasons, the tissue changes following acoronary thrombosis are divided into distinct zones. The central zone oftissue, i.e., usually that zone of tissue closest to the thrombus,becomes almost completely necrotic. This is surrounded by an area ofsevere ischemia. Outside this is an area of lesser ischemia called themarginal zone. Finally, there is a jeopardized zone which surrounds theentire area.

In studies with baboons, the central necrotic area was not affected byrecanalization of the vessel after several hours. However, muscle in theother zones which had undergone less severe damage during the ischemicperiod could be salvaged. A surprising finding was that lysing of thethrombus to produce a perfect arteriograph was insufficient to restorenormal flow in the majority of animals. (See Flameng, et al, J. Clin.Invest., Vol. 75, p. 84-90, 1985). Some further impediment to flow haddeveloped in the area supplied by the vessel during the time that it wasoccluded. In further studies, it was demonstrated that immediately afterremoving the obstruction to the vessel, the flow through the damagedtissue began at a high rate. However, within a short time the blood flowthrough the ischemic zone decreased and the tissue died.

Consequently, the regional blood flow immediately after reperfusion is apoor predictor of the salvage of myocardial tissue. If the blood flowthrough the damaged tissue remained near the normal levels, the successof tissue salvage was much greater. Hemorrhage occurred almostexclusively in the severely ischemic zone reflecting damage to the smallblood vessels. The hemorrhage, however, remained limited to the severlyischemic tissue and did not cause extension of the infarction or otherserious complication. Therapies which could preserve the blood flowthrough the small blood vessels distal to the major area of thrombusafter reperfusion could be expected to markedly increase the salvage ofmyocardial tissue.

The damage to heart muscle cells which occurs after lysing the thrombusis due to other factors as well as ischemia. Contact of fresh blood withdamaged or dead cells induces the influx of neutrophils, or pus cells,which can damage or kill heart cells which would otherwise haverecovered. Much of the damage caused by neutrophils has been attributedto superoxide ions. (For a general review, please see "Oxygen Radicalsand Tissue Injury" Proceedings of a Brook Lodge Symposium, AugustaMichigan, Barry Halliwell, Ed.). The superoxide anion can damage tissuein several ways. The interaction of the superoxide anion with hydrogenperoxide leads to the production of hydroxyl radicals which are highlytoxic and react rapidly with most organic molecules. Mannitol is aselective scavenger of hydroxyl radicals. The enzyme, superoxidedismutase, catalyzes the decomposition of the superoxide anion. Enzymessuch as superoxide dismutase, free radical scavengers or agents whichprevent the influx on neutrophils are able to increase the salvage ofheart muscle cells.

Continuing therapy is needed even after restoration of blood flow andsalvage of damaged tissue. The arteriosclerosis that caused the originalheart attack remains. American and European researchers have have foundthat arteriosclerosis still narrows the arteries in 70-80% of patientswhose clots were lysed by thrombolytic therapy. Many physicians believethis obstruction must be opened for long term benefits.

Balloon angioplasty is a procedure whereby a catheter with a smallballoon is inserted into the narrowed artery. The ballon is inflated,compresses the the atherosclerotic plaque against the vessel wall anddilates the artery. The effectiveness of this procedure is limited bythe effects of ischemia produced by the ballon, by embolization ofatheromatous material which lodges in distal vessels and by an increasedtendency for immediate or delayed thrombosis in the area damaged by theballoon. The ballon tears the tissue exposing underlying collagen andlipid substances which induce formation of thrombi. The thrombus mayocclude the vessel immediately or set up a sequence of events whichleads to occlusion many days or weeks later. In addition, there is aninterruption of blood flow to the heart tissue when the balloon isinflated. When the blood flow is interrupted, tissue downstream from theballoon is deprived of blood and can be damaged. Balloon angioplasty isrepresentative of numerous clinical and experimental procedures forrepairing the lumen of diseased arteries and vessels.

What is needed is a means of rendering the surface of the dilated vesselless thrombogenic, improving the blood flow through the distal tissueand breaking the embolized material into smaller pieces which are lesslikely to produce embolic damage. A means of restoring blood flowthrough the microcapillaries downstream from the site of ballooninflation is also required.

Another area where fibrinogen/fibrin plays a role is tumors. There isnow strong evidence that fibrinogen-related proteins are localized insolid tumors. The anatomical distribution of fibrin in tumors variesdepending on the tumor type. In carcinomas, fibrin is deposited in thetumor stroma and around tumor nests and may be particularly abundanttoward the tumor periphery and at the tumor host interface. By contrast,fibrin is often less prominent in older, more central tumor stromacharacterized by sclerotic collagen deposits. Fibrin may also be foundbetween individual carcinoma cells. In some, but not all such cases,interepithelial fibrin deposits are related to zones of tumor necrosis;however, zones of tumor necrosis are not necessarily sites of fibrindeposition. Fibrin deposition in sacomas has been less carefully studiedthan that in carcinomas. In lymphomas, fibrin deposits may be observedbetween individual malignant tumor cells as well as between adjacent,apparently reactive benign lymphoid elements. Fibrin has been reportedto appear in zones of tumor sclerosis, as in Hodgkin's disease. Researchhas indicated that the pattern and extent of fibrin deposition arecharacteristic for a given tumor. (See Hemostasis and Thrombosis, BasicPrinciples and Clinical Practice, "Abnormalities of Hemostasis inMalignancy", pp. 1145-1157, ed. by R. W. Colman, et al., J. B.Lippincott Company, 1987).

The lack of a uniform vascular supply to tumors can impede diagnosticand therapeutic procedures. For example, hypoxic tumors are lesssusceptible to many drugs and to radiation. Conventional drugs and newdrugs, such as monoclonal antibody conjugates, are not effective unlessthey are delivered to tumor cells. Fibrin deposits that surround sometypes of tumors inhibit delivery of the drugs to the tumor. The bloodsupply of tumors is further compromised by other factors as well. Bloodvessels in tumors are frequently small and tortuous. The hydrodynamicresistance of such channels further impedes the flow of blood to tumors.

Finally, lipid material on the atherosclerotic wall contributes to thebulk of the plaque which narrows the lumen of the artery and produces ahighly thrombogenic surface. What is needed is a method of extracting orcovering lipids from atherosclerotic plaques which leaves their surfacesless thrombogenic and reduces their bulk.

Use of copolymers prepared by the condensation of ethylene oxide andpropylene oxide to treat an embolus or a thrombus has been described(See U.S. Pat. No. 3,641,240). However, the effect is limited torecently formed, small (preferably microscopic) thrombi and emboli whichare composed primarily of platelets. To be effective, the compound mustbe used within 20 minutes after the initiation of thrombosis.

The use of the ethylene oxide and propylene oxide copolymer has littleor no effect on a clot in a patient who has suffered a severe coronaryinfarction because such patients almost never receive treatment within20 minutes following initiation of thrombosis. It is likely that manypersons do not develop symptoms until the thrombus reaches considerablesize. The clots that are occluding the blood vessel in these patientsare large and stable clots. Stable clots are clots in which the fibrinhas undergone cross linking. Fibrin which has undergone crosslinking isnot effected by presence of the ethylene oxide-propylene oxidecopolymers. The copolymers only affect new clots composed primarily ofplatelets in which the newly formed fibrin has not crosslinked.

Another problem that commonly occurs in damaged tissue where blood flowis interrupted is a phenomenon called "no reflow" phenomenon. This is aconditions wherein blood flow is interrupted to a tissue. When bloodflow is restarted, such as after a clot is removed, flow in the smallermicrocapillaries is often impaired because blood cells tend to clump inthe microcapillaries thereby inhibiting flow of blood to the tissue.This can result in damage to the tissue.

In addition, such a composition would be useful in removing clots fromsolid tumors, increasing flow through tortuous channels and therebyallow delivery of therapeutic drugs to the tumor.

A further need is a composition that can be used to prevent or treat "noreflow" phenomenon. Such a composition should be capable of causingblood to flow in tissue after blood flow has stopped thereby preventingtissue damage.

Increased demand for platelet concentrates to treat bleeding associatedwith thrombocytopenia has prompted the need to determine optimal methodsof storing platelets prior to transfusing them into a patient.

Viability, as measured by survival of ⁵¹ CR-labeled platelets, seemsbest preserved when stored at 22° C., whereas platelet function, asmeasured by the ability of platelets to aggregate in response toepinephrine, collagen, and adenosine diphosphate is better preserved at4° C. Platelets stored at room temperature for 48 to 72 hours as well asthose kept refrigerated for 24 to 48 hours have been found by differentinvestigators to produce satisfactory increases in platelet levels whentransfused to thombocytopenic patients.

Thus, blood blanks wishing to store platelets prior to their transfusioninto a patient are faced with the dilemma of whether they should be keptat room temperature, thus preserving their lifespan but possiblycompromising their functional capacity, or whether they should be storedin the approximately 4° C. with the resultant preservation of functionbut shortening of post-transfusion survival time.

What is needed is a composition and method which can be added to asuspension of platelets which will preserve both lifespan and functionof the platelets so that the platelet suspension can be stored forlonger periods of time. Such a composition should also be capable ofinhibiting the aggregation or clumping of platelets in the suspension.

Finally, the present inventor has identified a phenomenon calledpathological hydrophobic interactions between blood components and thosecells which line the blood vessels. This phenomenon is typicallyencountered when tissue is damaged in some manner. These pathologicalhydrophobic interactions cause blood flow to be reduced or stoppedthereby causing damage to surrounding tissue. What is needed is acomposition and method for reducing the pathological hydrophobicinteractions and thereby allowing blood to flow into the damaged tissue.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method is provided fortreating pathologic hydrophobic interactions in blood and otherbiological fluids. In particular, the method of the present inventionlimits or prevents damage due to (1) high concentrations of hydrophobicsoluble fibrin, (2) cell damage due to the exposing of hydrophobicdomains in the cell membrane that are usually hidden or buried. Themethod of the present invention also has a cytoprotective effect.

The method of the present invention increases flow of biological fluidsin diseased tissue. The flow in such tissue is commonly impeded becauseof the pathological hydrophobic interactions between cells and/orcertain molecules. The present invention includes the use of a surfaceactive copolymer for treatment of diseases and conditions in whichresistance to blood flow is caused by injury due to the presence ofadhesive proteins or damaged membranes. Such proteins and damagedmembranes increase resistance in the microvasculature by increasingfriction and reducing the effective radius of the blood vessel. The mostimportant of these proteins are fibrinogen and soluble fibrin.

The method comprises administering to an animal or human an effectiveamount of a surface active copolymer with the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably about1200 to 3500, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes approximately 50% to 95% by weightof the compound.

Also in accordance with the present invention, a fibrinolyticcomposition and method is provided that is effective in dissolving bloodclots and reestablishing and maintaining blood flow through thrombosedcoronary or other blood vessels. The fibrinolytic composition of thepresent invention comprises an enzyme, such as streptokinase, urokinase,prourokinase, tissue plasminogen activator, or other proteolytic enzyme,and a surface active copolymer. The surface active copolymer can be anethylene oxide-propylene oxide condensation product with the followinggeneral formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably about1200 to 3500, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes approximately 50% to 95% by weightof the compound.

The fibrinolytic composition of the present invention is usuallyadministered by intravenous injection into a patient but can beadministered by intramuscular or other parenteral injection.

The present invention provides a composition that can be administered topatients who have a blood clot occluding a blood vessel. The combinationof proteolytic enzyme and surface active copolymer according to thepresent invention increases blood flow around a clot, rapidly lyses aclot, and provides futher protection to the patient by preventing a newclot from forming and reducing reperfusion injury.

Because the fibrinolytic composition of the present invention stabilizesthe patient to a greater extent than treatments in the prior art, theadministration of more invasive procedures, such as balloon angioplasty,can be delayed thereby permitting selection of conditions for theinvasive treatment that are most favorable to the patient. In addition,the treatment of myocardial infarction by use of a proteolytic enzymesuch as t-PA or streptokinase can be delayed because the addition of thesurface active copolymer will limit the damage to the heart tissue.

Another embodiment of the present invention is a composition comprisingthe combination of the surface active copolymer and free radicalscavengers including but not limited to, superoxide dismutase andmannitol, mercaptopropionyl glycine. The surface active copolymer canalso be used with agents that prevent the generation of free radicalspecies including, but not limited to, ibuprofen, BW755C, nafazatrom,prostacyclin, iloprost, allopurinol, phenytoin as well as otheranti-inflammatory or cytoprotective drugs. It is to be understood thatthe term free radical scavengers includes both the scavenger compoundsand the compounds that prevent the generation of free radial species.The present invention includes a composition comprising the combinationof surface active copolymer, clot lysing enzyme and free radicalscavenger and also the a composition comprising combination of surfaceactive copolymer and free radical scavenger alone.

In accordance with the present invention, a composition and method isprovided that is effective in prolonging the function and lifespan ofplatelets in suspension. The method comprises adding an effective amountof a surface active copolymer to the platelet suspension. The surfaceactive copolymer can be an ethylene oxide-propylene oxide condensationproduct with the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, perferably about1200 to 3500, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes approximately 50% to 95% by weightof the compound.

The present invention also embodies a method for efficiently deliveringdrugs to and into diseased or damaged tissue. This includes tissuedamaged by infection, trauma, burns, or other noxious insult.

Accordingly, it is an object of the present invention to provide amethod of treating pathologic hydrophobic interactions of components inblood or other biological fluids.

It is a further object of the present invention to provide a method forprotecting cells during and after an ischemic period.

It is a further object of the present invention to provide a method andcomposition for protecting tissue after a burn.

It is yet another object of the present invention to provide a methodfor protecting myocardial cells, endothelial cells and other cells fromischemia.

It is another object of the present invention to provide a method ofenhancing the ability of cells and tissue to recover from ischemia.

It is an object of the present invention to provide a combination offibrinolytic enzymes with a surface active copolymer to produce asynergistic action in lysing blood clots. This combination can beformulated either with standard doses of enzyme to increase the rate orlikelihood of lysing a clot or at lower doses of enzyme to reduce sideeffects while maintaining efficacy for lysing clots.

It is another object of the present invention to provide a compositionthat will reduce the need for anticoagulation in therapy of thrombosisand thereby lessen the danger of hemorrhage.

It is another object of the present invention to provide a compositionthat accelerates the dissolution of clots by freeing aggregatedplatelets and blocking further platelets from aggregating to the clot.

It is yet another object of the present invention to provide acomposition that can reduce the dose of proteolytic enzyme required tolyse a clot and thereby reduce the incidence of complications.

It is another object of the present invention to provide a compositionthat contains a surface active copolymer and a free radical or oxygenscavenger, such as superoxide dismutase mannitol, and/ormercaptopropionyl glycine.

It is a further object of the present invention to provide a compositionthat can promote blood flow through microvascular channels of tissuedamaged by ischemia and reduce the amount of tissue which undergoesnecrosis.

It is a further object of the present invention to provide a method fordelivering drugs to damaged or diseased tissue.

It is a further object of the present invention to provide a compositionthat will significantly reduce the risk of rethrombosis after treatementwith fibrinolytic enzymes.

If is a further object of the present invention to provide a compositionthat will promote removal of lipids from atherosclerotic vessel wallsand thereby lessen the incidence of rethrombosis.

It is another object of the present invention to provide an improvedfibrinolytic composition that is capable of lysing fibrin depositsassociated with tumors.

It is another object of the present invention to provide a compositionwhich will increase blood flow through tortuous channels such as occurin tumors and during crisis of sickle cell disease.

It is another object of the present invention to provide an improvedcomposition and method for ex vivo preservation of organs.

It is another object of the present invention to provide a compositionthat will reduce the risk of rethrombosis and thereby allow delay inadministering balloon angioplasty or other invasive procedures fortreatment of the compromised vessels.

It is another object of the present invention to provide a compositionwhich will reduce the risk of thrombosis immediately or at some timeafter invasive procedures such as balloon angioplasty which damageendothelial cells of the vasculature.

It is a further object of the present invention to provide a compositionto block the aggregation of platelets in blood vessels distal to thethrombosis and thereby limit extension of tissue damage.

It is yet another object of the present invention to provide acomposition to improve blood flow through and around tissue withextensive necrosis of myocardial or other cells thereby retardingnecrosis of additional myocardial tissue.

It is another object of the present invention to provide a compositionwhich will reduce the influx of neutrophils into damaged tissue andthereby reduce the extent of injury caused by toxic products ofneutrophils.

It is yet another object of the present invention to provide acomposition that will decrease the amount of ischemia caused blockage ofblood flow by a thrombus.

It is yet another object of the present invention to provide a methodfor increasing blood flow in ischemic or damaged tissue thereby reducingdamage to the tissue.

It is another object of the present invention to provide a method fortreating burns.

It is a further object of the present invention to provide a combinationof a thrombolytic enzyme, balloon angioplasty or other operativeprocedures and a surface active copolymer to produce an improved methodof removing a thrombus or thrombogenic occlusion and reducingobstructive conditions which promote rethrombosis.

It is another object of the present invention to provide a compositionand method for the treatment of crisis in sickle cell disease.

It is another object of the present invention to provide a compositionthat is effective in restarting blood flow through microcapillariesafter ischemia.

It is an object of the present invention to provide composition andmethod for prolonging the life-span and function of platelets.

It is another object of the present invention to provide a compositionand method that will allow platelet suspensions to be stored for longerperiods of time then is presently possible with prior art methods.

It is another object of the present invention to provide a compositionand method that can be added to conventional platelet containers so thatplatelet suspensions can be stored for a longer period of time.

It is yet another object of the present invention to provide acomposition and method that can be used to prolong the lifespan of cellsuspensions.

It is yet another object of the present invention to provide a method ofstoring a concentrated suspensions of platelets whereby plateletfunction is prolonged thereby allowing longer storage times.

It is another object of the present invention to provide a method ofstoring a concentrated suspension of platelets for transfusion into apatient.

It is yet another object of the present invention to provide acomposition and method for treatment of shock using a surface activecopolymer with a plasma extender.

It is another object of the present invention to provide a method andcomposition for treating microvascular diseases caused by endotoxin suchas endotoxin shock or laminitis in horses.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the effect of the surface active copolymer ondissolving a clot with and without tPA.

FIG. 2 is a graph showing the effect of the surface active copolymer onflow of blood through a clot.

FIG. 3 illustrates the ability of the surface active copolymer topreserve platelet function when using ADP as the agonist.

FIG. 4 illustrates the preservation effect of the surface activecopolymer on platelets over 24 and 72 hours when using collagen as anagonist.

FIG. 5 illustrates the effect of the surface active copolymer on bloodviscosity in patients before open heart surgery.

FIG. 6 illustrates the effect of the surface active copolymer on bloodviscosity in patients 6 hours after open heart surgery.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS

In accordance with the present invention, a method is provided fortreating pathologic hydrophobic interactions in blood and otherbiological fluids of humans and animals. The present invention includesthe use of a surface active copolymer for treatment of diseases andconditions in which resistance to blood flow is pathologically increasedby injury due to the presence of adhesive hydrophobic proteins ordamaged membranes. This adhesion is produced by pathological hydrophobicinteractions and does not require the interaction of specific ligandswith their receptors. Such proteins and/or damaged membranes increaseresistance in the microvasculature by increasing friction and reducingthe effective radius of the blood vessel. It is believed that the mostimportant of these proteins is soluble fibrin.

The method according to the present invention comprises administering tothe animal or human suffering from a condition caused by a pathologicalhydrophobic interaction an effective amount of a surface activecopolymer. The surface active copolymer may be adminsitered as asolution by itself or it may be administered with another agent,including but not limited to, a fibrinolytic enzyme, an anticoagulant,or an oxygen radical scavenger.

The surface active copolymer in all embodiments of the present inventioncomprises the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably about1200 to 3500, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes approximately 50% to 95%, by weightof the compound.

The most preferred surface active copolymer for use in the method of thepresent invention is a copolymer having the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.B H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400.

The surface active copolymer of the present invention is effective inany condition where there is a pathological hydrophobic interactionbetween cells and/or molecules. These interactions are believed to becaused by (1) a higher than normal concentration of fibrinogen, (2)generation of intravascular or local soluble fibrin, especially highmolecular weight fibrin, (3) increased friction in the microvasculature,or (4) mechanical or chemical trauma to blood components. All of theseconditions cause an increase in pathological hydrophobic interactions ofblood components such as cells and molecules.

It is believed that fibrin, especially soluble fibrin, increasesadhesion of cells to one another, markedly icnreases friction in smallblood vessels and increases viscosity of the blood, especially at lowshear rates. The effects of the surface active copolymer of the presentinvention are believed to be essentially lubrication effects becausethey reduce the friction caused by the adhesion.

Although not wanting to be bound by the following hypothesis, it isbelieved that the present invention acts according to the followingmechanism: Hydrophobic interactions are crucial deteminants of biologicstructure. They hold the phospholipids together in membranes and proteinmolecules in their native configurations. An understanding of thebiology of the surface active copolymer is necessary to appreciate thebiologic activities of the compound. Water is a strongly hydrogenbonding liquid which, in its fluid state, forms bonds in all directionswith surrounding molecules. Exposure of a hydrophobic surface, definedas any surface which forms insufficient bonds with water, produces asurface tenhsion or lack of balance in the hydrogen bonding of watermolecules.

This force can be exceedingly strong. The surface tension of pure wateris approximately 82 dynes/cm. This translates into a force of severalhundred thousand pounds per square inch on the surface molecules.

As two molecules or particles with hydrophobic surfaces approach, theyadhere avidly. This adhesion is driven by the reduction in free energywhich occurs when water molecules transfer from the stressednon-hydrogen bonding hydrophobic surface to the non-stressed bulk liquidphase.

The energy holding such surfaces together, the work of adhesion, is adirect function of the surface tension of the particles (See: Adamson AWPhysical Chemistry of Surfaces, Fourth Edition, John Wiley & Sons, NewYork, 1982):

    W.sub.AB =γA+γB-γAB

where W_(AB) =work of adhesion or the energy necessary to separate onesquare centimeter of particle interface AB into two separate particlesγA and γB are the surface tensions of particle A and particle B, γAB theinterfacial tension between them.

Consequently, any particles or molecules in the circulation whichdevelop significant surface tensions will adhere to one anotherspontaneously. Such adhesion within membranes and macromolecules isnecessary to maintain their integrity. We use the term "normalhydrophobic interaction" to describe such forces. Under normalcircumstances, all cells and molecules in the circulation havehydrophilic non-adhesive surfaces. Receptors and ligands which modulatecell and molecular interactions are generally located on the mosthydrophilic exposed surfaces of cells and molecules where they are freeto move about in the aqueous media and to interact with one another.Special carrier molecules are necessary to transport lipids and otherhydrophobic substances in the circulation. In body fluids such as blood,nonspecific adhesive forces between mobile elements are extremelyundesirable. We term these "pathologic hydrophobic interactions" becausethey restrict movement of normally mobile elements and promoteinappropriate adhesion of cells and molecules.

In damaged tissue, hydrophobic domains normally located on the interiorof cells and molecules may become exposed and produce pathologicadhesive surfaces whose interaction compounds the damage. Fibrindeposited along vessel walls also provide an adhesive surface. Suchadhesive surfaces appear to be characteristic of damaged tissue. It isbelieved that the ability of the surface active copolymer to bind toadhesive hydrophobic surfaces and convert them to non-adhesive hydratedsurfaces closely resembling those of normal tissues underlies itspotential therapeutic activities in diverse disease conditions.

Adhesion due to surface tension described above is different from theadhesion commonly studied in biology. The commonly studied adhesion isdue to specific receptor ligand interactions. In particular, it isdifferent from the receptormediated adhesion of the fibrinogen--vonWillibrands factor family of proteins (See generally Hemostasis andThrombosis, Basis Principles and Clinical Practice, ed. by Colman, etal., J.B. Lippincott Company (1987)).

Both the hydrophilic and hydrophobic chains of the surface activecopolymer have unique properties which contribute to biologic activity.The hydrophilic chains of POE are longer than those of most surfactantsand they are flexible. They bind water avidly by hydrogen bond acceptorinteractions with ether-linked oxygens. These long, strongly hydratedflexible chains are relatively incompressible and form a barrier tohydrophobic surfaces approaching one another. The hydroxyl moieties atthe ends of the molecule are the only groups capable of serving ashydrogen bond donors. There are no charged groups.

This extremely limited repertoire of binding capabilities probablyexplains the inability of the molecule to activate host mediator andinflammatory mechanisms. The POE chains are not necessarily inert,however. Polyoxyethylene can bind cations by ion-dipole interactionswith oxygen groups. The crown polyethers and reverse octablock copolymerionophores are examples of such cation binding (See Atkinson, TP, etal., Ion transport mediated by copolymers composed of polyoxyethyleneand polyoxypropylene. Am J Physiol 254;C20, 1988). It is possible thatthe flexible POE chains form configurations which bind and modulatecalcium and other cation movements in the vicinity of damaged membranesor othe hydrophobic structures.

The hydrophobic component of the surface active copolymer is large, weakand flexible. The energy with which it binds to a cell membrane orprotein molecule is less than the energy which holds the membranephospholipids together or maintains the tertiary conformation of theprotein. Consequently, unlike common detergents which dissolve membranelipids and proteins, the surface active copolymer adheres to damagedspots on membranes and prevents propagation of the injury.

The ability of the surface active copolymer to block adhesion offibrinogen to hydrophobic surfaces and the subsequent adhesion ofplatelets and red blood cells is readily demonstrated in vitro. Mostsurfactants prevent adhesion of hydrophobic particles to one another,however, the surface active copolymer has a unique balance of propertieswhich optimize the anti-adhesive activity while minimizing toxicity.Thus, the surface active copolymer is not routinely used by biochemistswho use nonionic surfactants to lyse cells or dissolve membraneproteins. The surface active copolymer protects cells from lysis. Thehydrophobe effectively completes with damaged cells and molecules toprevent pathologic hydrophobic interactions, but cannot disrupt the muchstronger normal hydrophobic interactions which maintain structuralintegrity.

The viscosity of blood is generally assumed to be the dominantdeterminant of flow through vessels with a constant pressure andgeometry. In the smallest vessels, however, those in damaged tissue,other factors become significant. When the diameter of the vessel isless than that of the cell, the blood cell must deform in order to enterthe vessel and then must slide along the vessel wall producing friction.The deformability of blood cells entering small vessels has beenextensively studied (See: Brooks DE and Evans EA Rheology of bloodcells, in Clinical Hemorheology. Applications in Cardiovascular andHematological Disease, Diabetes, Surgery and Gynecology. S Chien, JDormandy, E Ernst, and A Matrai, eds, Martinus Nijhoff Publishers,Dordrecht, 1987), but the adhesive or frictional component has not. Theadhesion of cells to vessel walls is generally attributed to specificinteractions with von Willebrand's factor and other specific adhesivemolecules (See: Thompson AR and Harker LA Manual of Hemostasis andThrombosis, Edition 3, F A Davis Company, Philadelphia, 1983). Our datasuggests that in pathologic situations, friction resulting fromnonspecific physicochemical adhesion between the cell and the vesselwall becomes a major determinant of flow.

Mathematically, both the strength of adhesion between two particles andthe friction force which resists sliding of one along the other aredirect functions of their surface tensions which are largely determinedby their degree of hydrophobic interaction. The friction of a cellsliding through a small vessel consists of an adhesion component and adeformation component (Lee LH Effect of surface energetics on polymerfriction and wear, in Advances in Polymer Friction and Wear, PolymerScience and Technology, volume 5A. L. H Lee, editor, Plenum Press, NewYork, 1974) which are in practice difficult to separate:

    F=Fa+Fd

where F is the friction of cells, Fa is the adhesion component and Fd isthe deformation component.

The deformation component within a vessel differs from that required forentry into the vessel. It may be similar to that which occurs in largervessels with blood flowing at a high rate of shear (Brooks and Evans,1987). Friction within blood vessels has been studied very little, butundoubtedly involves the same principles which apply to polymer systemsin which the friction force correlates directly with the work ofadhesion (Lee, 1974):

    Fa=kWA+c

where Fa is the adhesional component of the friction force, WA the workof adhesion, and k and c constants which pertain to the particularsystem studied. Many lubricants act as thin films which separate the twosurfaces and reduce adhesion (See: Adamson, 1982).

The effect of the surface active copolymer on microvascular blood flowwere evaluated in several models ranging from artificial in vitrosystems where critical variables could be rigidly controlled to in vivosystems mimicking human disease. First, the surface active copolymer canbe an effective lubricant when used at therapeutic concentrations in amodel designed to simulate movement of large cells through smallvessels. It markedly reduced the adhesive component of friction, but hadno detectable effect on the deformation component of friction. Second,the surface active copolymer greatly accelerates the flow through thenarrow channels formed by the thrombogenic surfaces of glass and air. Adrop of blood was placed on a cover slip and viewed under a microscopewith cinemicroscopy during the time it took the blood to flow to theedges of the cover slip in response to gentle pressure. The surfaceactive copolymer inhibited the adhesion of platelets to the glass andmaintained the flexibility of red cells which enabled them to passthrough the microscopic channels. While the surface active copolymer didnot inhibit the formation of rouleaux by red cells, it did cause therouleaux to be more flexible and more easily disrupted. Third, thesurface active copolymer increases the flow of blood through tortuouscapillary-sized fibrin-lined channels by over 20-fold (See Example IXherein). It decreased viscosity of the blood by an amount (10%) far toosmall to account for the increased flow.

In a more physiologic model, the surface active copolymer increasedcoronary blood flow by a similar amount in isolated rat hearts perfusedwith human red blood cells at a 30% hematocrit following ischemic damage(See Example X herein).

In an in vivo model of stroke produced by ligature of the middlecerebral artery of rabbits, the surface active copolymer increases bloodflow to ischemic brain tissue. As much as two-fold increase was measuredby a hydrogen washout technique. In each of these models, there werecontrols for hemodilution and there was no measurable effect onviscosity at any shear rate measured.

It is believed that available data suggests that the surface activecopolymer acts as a lubricant to increase blood flow through damagedtissues. It blocks adhesion of hydrophobic surfaces to one another andthereby reduces friction and increases flow. This hypothesis isstrengthened by the observation that the surface active copolymer haslittle effect on blood flow in normal tissues where such frictionalforces are small (See: Grover FL, Kahn RS, Heron MV, and Paton BC, Anonionic surfactant and blood viscosity. Arch Surg 106:307, 1973.)

The surface active copolymers of the present invention are notmetabolized by the body and are quickly eliminated from the blood. Thehalf-life of the copolymer in the blood is believed to be approximatelytwo hours. It is to be understood that the surface active copolymer inthe improved fibrinolytic composition of the present invention is notcovalently bound to any of the other components in the composition noris it covalently bound to any proteins.

The surface active copolymer can be administered with a fibrinolyticenzyme, a free radical scavenger, or it can be administered alone fortreatment of certain circulatory conditions which either are caused byor cause pathological hydrophobic interactions of blood components.These conditions include, but not limited to, myocardial infarction,stroke, bowel or other tissue infarctions, malignancies, adultrespiratory distress syndrome (ARDS), disseminated intravascularcoagulation (DIC), diabetes, unstable angina pectoris, hemolytic uremicsyndrome, red cell fragmentation syndrome, heat stroke, retained fetus,eclampsia, malignant hypertension, burns, crush injuries, fractures,trauma producing shock, major surgery, sepsis, bacterial, parasitic,viral and rickettsial infections which promote activation of thecoagulation system, central nervous system trauma, and during andimmediately after any major surgery. It is believed that treatment ofthe pathological hydrophobic interactions in the blood that occurs inthese conditions significantly reduces microvascular and othercomplications that are commonly observed.

The surface active copolymer of the present invention is also effectivein increasing the collateral circulation to undamaged tissues withcompromised blood supply. Such tissues are frequently adjacent to areasof vascular occlusion. The mechanism appears to be reducing pathologicalhydrophobic interactions in small blood vessels. Circulatory conditionswhere the surface active copolymers are effective include, but are notlimited to, cerebral thrombosis, cerebral embolus, myocardialinfarction, unstable angina pectoris, transient cerebral ischemicattacks, intermittent claudication of the legs, plastic andreconstructive surgery, balloon angioplasty, peripheral vascularsurgery, orthopedic surgery, especially when using a tourniquet.

The surface active copolymer has little effect on the viscosity ofnormal blood at shear rates ranging from 2.3 sec⁻¹ (low) to 90 sec⁻¹(high). However, it markedly reduces the abnormally high viscosity foundin postoperative patients and in those with certain pathologicconditions. This observation posed two questions: (1) what caused theelevated whole blood viscosity in these patients and, (2) whatmechanisms did the surface active copolymer, which has only minoreffects on the blood viscosity of healthy persons, normalize pathologicelevations in viscosity?

It is generally accepted that hematocrit and plasma fibrinogen levelsare the major deteminants of whole blood vicosity. This has beenconfirmed in normal individuals and in many patients with inflammatoryconditions. However, these factors could not explain the changes thatwere observed. In patients having coronary artery cardiac bypasssurgery, it was found that hematocrit fell an average of 23±4% andfibrinogen fell 48±9% within six hours after surgery. The viscosity didnot decrease as expected, but increased from a mean of 23±2 to 38±4centipoise (at a shear rate of 2.3 sec⁻¹). Viscosities in excess of 100were found in some patients. The abnormally high viscosity of blood wasassociated with circulating high molecular weight polymers of solublefibrin (See: Papadea C and Hunter R Effect of RheothRx™ copolymer onblood viscosity related to fibrin(ogen) concentration. FASEB J 2:A384,1988). The soluble fibrin levels rose from 19±5 μg/ml to 43±6 μg/mlduring surgery. These studies utilized a colorimetric enzymatic assayfor soluble fibrin (Wiman, B. and Rånby, M., Determination of solublefibrin in plasma by a rapid and quantitative spectrophotometric assay.Thromb. Haemost 55:189, 1986) and Western blotting procedures with SDSagarose gels to determine the molecular weight of the large proteinpolymers (See: Connaghan DG, Francis CW, Lane DA, and Marder VJ Specificidentification of fibrin polymers, fibrinogen degradation products, andcrosslinked fibrin degradation products in plasma and serum with a newsensitive technique. Blood 65:589, 1985).

In the absence of specific receptors, cells and molecules in thecirculation adhere to one another if the adherence reduces the freeenergy or surface tension between them. An assessment of the surfacetension of various components of the blood can be made by measuringcontact angles.

Red blood cells, lymphocytes, platelets, neutrophils all have contactangles in the range of 14 to 17 degrees. Peripheral blood proteins, suchas albumin, α₂ macroglobulin, and Hageman factor have contact angles inthe slightly lower range of 12-15. This means that these proteins haveno adhesive energy for the cells. In contrast, fibrinogen has a contactangle of 24 degrees and soluble fibrin of 31. Consequently, fibrinogenadheres weakly to red blood cells and other cells in the circulationpromoting rouleaux formation. Fibrin promotes a very much strongeradhesion than fibrinogen because its elevated contact angle and itstendency to form polymers with fibrinogen. Soluble fibrin in thecirculation produces the increased adhesion which results in a verymarkedly increased viscosity at low shear rates. This adhesion alsoinvolves the endothelial walls of the blood vessels. If the adhesiveforces are insufficient to slow movement of cells, they produce anincreased friction. This is especially important in the very small bloodvessels and capillaries whose diameters are equal to or less than thatof the circulating cells. The friction of cells sliding through thesesmall vessels is significant. The surface active copolymer of thepresent invention blocks the adhesion of fibrinogen and fibrin tohydrophobic surfaces of cells and endothelial cells. This prevents theiradhesion and lubricates them so there is a greatly reduced resistance toflow. This can be measured only partially by measurements of viscosity.

Whether a certain fibrinogen level is sufficient to cause a problem incirculation is dependent upon several parameters of the individualpatient. High hematocrits and high levels of fibrinogen are widelyregarded as the primary contributors to increased viscosity. However,elevated fibrinogen levels are frequently associated with elevatedsoluble fibrin in the circulation. Careful studies have demonstratedthat the fibrin is frequently responsible for the most severe changes.The normal level of fibrinogen is 200-400 μg/ml. It has been determinedthat, in most patients, fibrinogen levels of greater than approximately800 μg/ml will cause the high blood viscosity at the low shear ratesmentioned hereinabove. The normal level of soluble fibrin has beenreported to be approximately 9.2±1.9 (Wiman, B. and Rånby, M.,Determination of soluble fibrin in plasma by a rapid and quantitativespectrophotometric assay. Thromb. Haemost 55:189, 1986). Using the Wimanand Rånby assay, viscosity at low shear rates was unacceptably highabove about 15 μg/ml. It must be understood that soluble fibrin meansmolecular species that have a molecular weight of from about 600,000 toseveral million.

Numerous methods have been used for demonstrating soluble fibrin. Theseinclude cryoprecipitation especially cryofibrinogen. Heparin has beenused to augment the precipitate formation. Ethanol and protamine alsoprecipitate fribrin from plasma. Modern techniques have demonstratedthat the soluble fibrin in the circulation is generally complexed withsolubilizing agents. These are most frequently fibrinogen or fibrindegradation products. Des AA fibrin in which only the fibrin of peptideA moieties have been cleaved, tends to form relatively small aggregatesconsisting of one molecule of fibrin with two of fibrinogen. If both theA and B peptides have been cleaved to produce des AABB fibrin, then muchlarger aggregates are produced in the circulation. Fibrin degradationproducts can polymerize with fibrin to produce varying size aggregatesdepending upon the particular product involved.

Soluble fibrin in the circulation can markedly increase blood viscosity,especially at low shear rates. However, the relevance of this forclinical situations remains unclear. Viscosity assesses primarily theaggregation of red blood cells which is only one of many factors whichdetermine in vivo circulation. Other factors affected by soluble fibrinare the endothelial cells, white blood cells and platelets. Solublefibrin is chemotactic for endothelial cells, adheres to them avidly andcauses their disorganization. It also has stimulatory effects for whiteblood cells especially macrophages. Some of the effects of solublefibrin may be mediated by specific receptors on various types of cells.However, since the free energy, as measured by contact angles of solublefibrin, is less than that of any other plasma protein, it adheres avidlyby a nonspecific hydrophobic interactions to virtually all formedelements in the blood.

Circulating soluble fibrin is normally cleared by macrophages andfibrinolytic mechanisms without producing damage. However, if theproduction of soluble fibrin is too great or if the clearance mechanismhave been compromised or if complicating disease factors are present,then soluble fibrin can induce deleterious reactions.

Soluble fibrin is produced in damaged or inflamed tissues. Consequently,its effects are most pronounced in these tissues where it coatsendothelial cells and circulating blood cells in a fashion whichmarkedly reduces perfusion. The largest effects are in the small bloodvessels where soluble fibrin coating the endothelial cells and whiteblood cells produces a severe increase in friction to the movement ofwhite cells through the small vessels. Friction appears to be a muchmore severe problem with white blood cells and red blood cells becausethey are larger and much more rigid.

If production of soluble fibrin is sufficient, then effects are noticedin other areas. The best studied is the adult respiratory distresssyndrome where soluble fibrin produced in areas of damaged tissueproduces microthrombi and other processes in the lungs which can causepulmonary failure. However, lesser degrees of vascular compromise can bedemonstrated in many other organs.

Soluble fibrin, either alone or in complex with fibrinogen and othermaterials is now recognized as being a major contributor to thepathogenesis of a diverse range of vascular diseases ranging fromcoronary thrombosis through trauma, burns, reperfusion injury followingtransplantation or any other condition where there has been localized orgeneralized activation of coagulation. A recent study demonstrated thatvirtually all patients with acute myocardial infarction or unstableangina pectoris have markedly elevated levels of soluble fibrin in theircirculation.

An example of the effects of soluble fibrin has been shown in thestudies using dogs. A normal dog is subjected to a hysterectomy. Then,while the animal is still under anesthesia, the external jugular vein iscarefully dissected. Alternatively, the vein may be occuled by gentlepressure with the fingers for seven minutes. It is examined by scanningelectron microscopy for adhesion of fibrin, red blood cells and otherformed elements.

One finds that very few cells adhere to the endothelia of veins fromdogs which had not undergone hysterectomy, whether or not there had beenstasis produced by seven minutes occlusion. Similarly, there was only asmall increase in adhesion of red blood cells to the endothelium of thejugular vein in animals who had undergone hysterectomy. If, however, theanimals had a hysterectomy in addition to mild seven minute occlusion ofthe veins, then there was a striking increase in adhesion of formedelements of blood to the endothelial surfaces in some cases producingfrank mural thrombi. Both red blood cells and fibrin were visiblyadherent to the endothelial surfaces. In addition, there was disruptionof the normal endothelial architecture. All of the animals had elevatedlevels of soluble fibrin after the surgery. This model demonstrates theeffects of soluble fibrin produced by relatively localized surgery toproduce a greatly increased risk of deep vein thrombosis at a distantsite.

The surface active copolymer of the present invention addresses theproblems of fibrin and fibrinogen in the blood by inhibiting theadhesion of fibrin, fibrinogen, platelets, red blood cells and otherdetectable elements of the blood stream. It blocks the formation of athrombus on a surface. The surface active copolymer of the presentinvention has no effect on the viscosity of water or plasma. However, itmarkedly increases the rate of flow of water and plasma in smallsegments through tubes. The presence of air interfaces at the end of thecolumns or air bubbles which provide a significant surface tensionproduce a friction along the walls of the tubes. The surface activecopolymer of the present invention reduces this surface tension and thefriction and improves flow. This is an example whereby the surfaceactive copolymer of the present invention improves flow of fluid throughtissues through a tube even though it has no effect on the viscosity ofthe fluid as usually measured.

The surface active copolymer of the present invention has only a smalleffect on the viscosity of whole blood from normal individuals. It haslittle effect on the increase that occurs within high hematocrit.However, it has an effect on the very large increase in viscosity at lowshear rates thought to be caused by soluble fibrin and fibrinogenpolymers.

Recent studies demonstrate that the surface active copolymer also hasthe ability to protect myocardial and other cells from a variety ofnoxious insults. During prolonged ischemia, myocardial cells undergo"irreversible injury." Cells which sustain irreversible injury aremorphologically intact but are unable to survive when returned to anormal environment. Within minutes of reperfusion with oxygenated blood,cells containing such occult lesions develop swelling and contractionbands and die.

Irreversibly injured myocardial cells have mechanical and osmoticfragility and latent activation of lipases, proteases and other enzymes.Reperfusion initiates a series of events including calcium loading, cellswelling, mechanical membrane rupture and the formation of oxygen freeradicals which rapidly destroy the cell. The surface active copolymerretards such injury in the isolated perfused rat heart model. Themechanisms probably include osmotic stabilization and increasedmechanical resistance in a fashion similar to that known for red bloodcells.

The protective effects of the surface active copolymer on the myocardiumare not limited to the myocardial cells. It also protects theendothelial cells of the microvasculature as assessed morphologically.By maintaining the integrity of such cells and helping to restore andmaintain non-adhesive surfaces, the surface active copolymer tends toreduce the adhesion of macromolecules and cells in the microvasculature,to reduce coronary vascular resistance and to retard development of theno reflow phenomenon.

Examples of conditions where the present invention can be used is in thetreatment of sickle cell disease and preservation of organs fortransplantation. In both of these embodiments, blood flow is reducedbecause of pathologic hydrophobic interactions.

During a sickle cell crisis, sickled red blood cells aggregate becauseof the abnormal shape of the cells. In many cases, there are highconcentrations of soluble fibrin due to disseminated intravascularcoagulation. This results pathological hydrophobic interactions betweenblood cells, cells lining the blood vessels and soluble fibrin andfibrinogen. By administering to the patient the surface active copolymerembodied in the present invention, blood flow is increased and tissuedamage is thereby reduced. It is contemplated as part of the presentinvention that the surface active copolymer may be given prior to asickle cell crisis to prevent onset of the crisis. In addition, thesolution with the effective amount of surface active copolymer may alsocontain an effective amount of anticoagulant.

In organs that have been removed from a donor for transplantation, thetissue is damaged due to ischemia and lack of blood. Preferably, thesurface active copolymer is mixed with a perfusion medium. The perfusionmedia that can be used with the surface active copolymer are well knownto those of ordinary skill in the art. The perfusion media can also bewhole blood or plasma. The solution can be perfused through the organthereby reducing the damage to the tissue. Because the tissue damage isreduced by perfusing the organ with the surface active copolymersolution, the time the organ is viable and therefore the time the organcan be transplanted is increased.

Because the surface active copolymer improves flow of blood throughdiseased or damaged tissue with minimal effect on blood flow in normaltissue, it is contemplated that the present invention includes a methodfor delivering drugs to damaged tissue comprising the step ofadministering to the animal or human a solution containing:

an effective amount of a drug; and

an effective amount of a surface-active copolymer with the followinggeneral formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, and b is aninteger such that the hydrophile portion represented by (C₂ H₄ O)constitutes approximately 50% to 95% by weight of the compound.

Any drug that has an activity in diseased or damaged tissue is suitablefor this embodiment of the present invention. These drugs include

1. Antimicrobial drugs

antibiotics

antifungal drugs

antiviral drugs

antiparasitic drugs

2. antifungal drugs;

3. chemotherapeutic drugs for treating cancers and certain infections;

4. free radical scavenger drugs including those drugs that prevent theproduction of free radicals;

5. fibrinolytic drugs;

6. perfusion media;

7. antiinflammatories, including but not limited to both steroids andnonsteriod antiinflammatory drugs

8. membrane stabilizers such as dilantin;

9. anticoagulants;

10. ionotropic drugs such as calcium channel blockers;

11. autonomic nervous system modulators;

Solutions which may be employed in practicing the present inventioninclude, but are not limited to, saline (a solution of sodium chloride,containing approximately 8.5 to 9.5 grams of sodium chloride in 1000 ccof purified water), Ringer's solution, lactated Ringer's solution,Krebs-Ringer's solution, and various sugar solutions. All of thesesolutions are well known to one of ordinary skill in the art. Otherisotonic solutions can be used to prepare a solution of the surfaceactive copolymer. However, it is to be understood that the presentinvention may be administered as a solution that is not isotonic. Thesurface active copolymer can be administered in a non-aqueous solution.

The method for treating pathologic hydrophobic interactions of thepresent invention includes administering the solution of surface activecopolymer by intravenous injection. However, it is to be understood thatthe solution of surface active copolymer can be administered byintramuscular, subcutaneous parenteral or any other route of injection.It is contemplated as part of the present invention that the surfaceactive copolymer could be administered orally either with an agent thatpromotes absorption of the copolymer by the gastrointestinal tract or bythe surface active copolymer itself. In addition, the surface activecopolymer can be administered transdermally.

The final concentration of surface active copolymer in blood or otherbiologic fluids used to practice the present invention is betweenapproximately 0.01 and 10 mg/ml. The preferred concentration of surfaceactive copolymer used to practice the present invention is betweenapproximately 0.1 and 2 mg/ml with the most preferred concentrationbetween approximately 0.4 and 0.8 mg/ml of fluid.

The present invention also comprises a composition which lyses bloodclots and reestablishes and maintains blood flow through a thrombosedcoronary vessel or other blood vessel. The fibrinolytic composition ofthe present invention is a solution containing an effectiveconcentration of a proteolytic enzyme and an effective concentration ofa surface active copolymer. The combination of the two components issurprisingly effective in dissolving blood clots that are blocking bloodvessels. In addition, the fibrinolytic composition of the presentinvention is highly effective in preventing a blood clot from reformingand in maintaining blood flow through the blood vessel and affectedischemic tissue.

The present invention also encompasses the combination of an effectiveamount of surface active copolymer and an effective amount of freeradical scavenger including, but not limited to, superoxide dismutase(SOD), mannitol, or mercaptopropionyl glycine or a combination of two ormore of the compounds. The combination of the two substances has beenshown to increase the flow of blood through ischemic tissue. Inparticular, the combination of surface active copolymer and SOD ormannitol has been shown to increase tissue survival after occlusion ofblood flow to the tissue (See Example VII and VIII herein).

It is thought that the fibrinolytic composition of the present inventionimproves the flow of blood through narrow passages around clots andthereby increases the delivery of the proteolytic enzyme to the clot.The present invention also speeds the rate of dissolution of the clot bythe enzyme and increases the proportion of clots lysed by promotingdelivery of enzyme to clots which would not otherwise be exposed tosufficient enzyme for their dissolution. In addition, the fibrinolyticcomposition of the present invention reduces the dose of fibrinolyticenzyme required for particular applications and thereby reduces theincidence of complications due to side effects caused by the enzymes.

The fibrinolytic composition of the present invention reduces the riskof immediate rethrombosis by accelerating the dissolution of clots andfreeing aggregated platelets and blocking further platelets fromaggregating to the clot or clot site. By reducing the risk of immediaterethrombosis, the fibrinolytic composition of the present invention willallow delay of balloon angioplasty or other invasive procedures fortreatment of the compromised vessels which have become thrombosed. Thedelay will permit selection of conditions for invasive treatment mostfavorable to the patient.

Solutions which may be employed in the preparation of the fibrinolyticcomposition of the present invention include, but are not limited to,saline (a solution of sodium chloride, containing approximately 8.5 to9.5 grams of sodium chloride in 1000 cc of purified water), Ringer'ssolution, lactated Ringer's solution, Krebs-Ringer's solution, andvarious sugar solutions. All of these solutions are well known to one ofordinary skill in the art. However, it is to be understood that thefibrinolytic composition of the present invention may be administered asa solution that is not isotonic.

The present invention includes use of the surface active copolymer withan effective amount of anticoagulant to permit blood flow throughischemic tissue. Anticoagulants that can be used with the presentinvention include, but are not limited to, heparin, low molecular weightheparin, coumarin derivatives, and warfarin. It is to be understood thatthe surface active copolymer of the present invention could be used withany one anticoagulant or with a combination of anticoagulants. It isalso understood that the concentration of anticoagulant to be used withthe surface active copolymer is well known to those of ordinary skill inthe art. It has been found that administration of the surface activecopolymer with anticoagulants increases blood flow through the ischemictissue in a synergistic manner (See Example VI herein).

The surface active copolymer is preferably an ethylene oxide-propyleneoxide condensation product with the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably from1750 to 3500, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes from about 50% to 95% by weight ofthe compound. These copolymers are sold under the general trademark ofPluronic® polyols and are available from the BASF Corporation(Parsippany, N.J.). The preferred formula of the present invention issold under the trademark RheothRx® copolymer and is available fromCytRx® Corporation (Norcross, Ga.).

The present invention also includes A method for preventing blockage incatheters comprising adding an effective amount of a surface activecopolymer to the fluid being delivered through the catheter, saidsurface active copolymer comprising the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, and b is aninteger such that the hydrophile portion represented by (C₂ H₄ O)constitutes approximately 50% to 95% by weight of the compound. It iscontemplated that the surface active copolymer can be used to maintainthe catheter over long periods of time. The method of the presentinvention can be used to maintain the patency of catheters and dialysismaterials in hemodialysis, peritoneal dialysis, intravascular catheters,bladder catheters and central nervous catheters.

The preferred concentration of the surface active copolymer of thepresent invention for use in maintaining catheters and the like isbetween approximately 0.01 mg/ml and 10 mg/ml with a preferredconcentration of between 0.1 mg/ml and 2 mg/ml. The method of thepresent invention blocks adhesion of proteins to catheters that areimplanted intraperitoneally, intrapleurally or in any body cavitythereby reducing the potential for infection.

The present invention also provides a composition and method for storingconcentrated platelet suspensions in a bag or other container. Thepresent invention allows the platelet suspension to be stored either atroom temperature or at refrigerator temperatures for longer periods oftime than possible with prior art methods while still maintaining theplatelets in a state where they are useful for transfusion into apatient. This state includes retention of platelet function andmorphology.

Platelets suspensions treated according to the present invention can bestored in conventional plastic bags normally used to store platelets.Experiments with human platelet show that these platelet suspensionstreated according to the present invention do not aggregatespontaneously as much as untreated platelets. Treated platelets retaintheir ability to aggregate in response to various stimuli such asadenosine diphosphate (ADP), thrombin, collagen, and epinephrine for amuch longer time than untreated platelets.

The method of storing platelets according to the present inventionincludes adding an effective amount of a surface active copolymer to asuspension of platelets and mixing briefly to disperse the copolymerthroughout the platelet suspension. Alternatively, the copolymer can beadded to the platelet container before adding the platelet suspension.In this way, the containers can be supplied to the blood bank or otherlocation where blood is processed and the platelet suspension can beadded to the container with the copolymer. The container with theplatelet suspension therein can then be stored until the platelets areto be used.

Prior art methods of storing platelet suspensions have been largelyunsatisfactory. Platelet function, as measured by the ability ofplatelets to respond to aggregation stimuli, in platelet suspensionsstored at room temperature is rapidly lost. When platelets are storedaccording to the present invention, platelets can be stored for longerperiod of time and retain platelet function. The preferred concentrationof the surface active copolymer of the present invention for use inpreserving platelets is between approximately 0.01 mg/ml and 10 mg/mlwith a preferred concentration of between 0.1 mg/ml and 2 mg/ml.

Another embodiment of the present invention is an improved plasmaextender composition and method of use. The improved plasma extender ofthe present invention comprises a conventional plasma extender and aneffective amount of a surface active copolymer with the followinggeneral formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably from1750 to 3500, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes from about 50% to 95% by weight ofthe compound.

The plasma extenders that can be used with the present inventioninclude, but are not limited to, various dextran solutions, hydroxyethylstarch, and albumin and both natural and fixed or stabilized hemoglobin.The preferred concentration of the surface active copolymer of thepresent invention for use with plasma extenders is between approximately0.01 mg/ml and 10 mg/ml with a preferred concentration of between 0.1mg/ml and 2 mg/ml. The most preferred concentration of the surfaceactive copolymer is approximately 0.6 mg/ml.

The concentration of surface active copolymer contemplated in thepresent invention can vary depending on the total volume of solutionneeded in the particular circumstances. The total amount of blockcopolymer employed in the present invention will also vary depending onthe size and type of thrombus or embolus, the particular copolymeremployed, the particular proteolytic enzyme employed, and the size andweight of the patient.

The copolymer can be used over a wide range of concentrations with nosevere adverse side effects. It is believed that the copolymer israpidly excreted intact; as much as 90% of the copolymer administered isexcreted within three hours. Because of its low toxicity and the rapidclearance from the body, the copolymer can be administered over a longperiod of time.

The surface active copolymer of the present invention may be employed byadmixing with blood in any standard manner. Preferably, however, thesolutions are intravenously injected into the blood stream either as abolus, slow drip or a combination of both. The solutions are generallyadmixed with the blood in a manner so as to maintain a substantiallysteady venous pressure.

It is to be understood that separate administration of a solution of thesurface active copolymer and a fibrinolytic enzyme or other agent arecontemplated in the present invention. For example, a solution of thesurface active copolymer and a solution of a fibrinolytic enzyme couldbe prepared separately and administered simultaneously or sequentiallyto a patient suffering from a thrombus blocking a coronary artery.Simultaneous or sequential administration of the two components(copolymer and fibrinolytic enzyme) of the fibrinolytic composition ofthe present invention has the same effect as administering thecomponents together and is therefore contemplated in the presentinvention.

The proteolytic enzymes that can be used in the fibrinolytic compositionof the present invention include, but are not limited to, streptokinase(available from Hoechst-Roussel under the trademark Streptase®),urokinase (available from Abbot Laboratories, North Chicago, Ill. underthe trademark Abbokinase®) and tissue plasminogen activator andActivase™ (Genentech, South San Francisco, Calif.). The tissueplasminogen activator can be derived from eukaryotic cells such as humanmelanoma cells or can be made by genetic engineering methods. Some ofthe fibrinolytic enzymes are only sparingly soluble in water and musttherefore be emulsified with the surface active copolymer beforeadministration to the patient.

Ideally, a bolus injection of the copolymer solution without the enzymeis administered before the present invention is administered. Forexample, a 3% solution of the copolymer in 5% isotonic dextrose isinjected within a two minute period so that the blood concentration ofcopolymer is approximately 0.6 mg/ml. In addition, it can beadvantageous to administer a solution of the copolymer by intravenousdrip at a rate of about 25 mg/kg body weight/hour to obtain of bloodconcentration of the copolymer of approximately 0.6 mg/ml for up to fourdays or longer following the administration of the fibrinolyticcomposition of the present invention. This treatment will aid inpreventing a clot from reforming.

The surface active copolymer blocks are formed by condensation ofethylene oxide and propylene oxide at elevated temperature and pressurein the presence of a basic catalyst. There is some statistical variationin the number of monomer units which combine to form a polymer chain ineach copolymer. The molecular weights given are approximations of theaverage weight of copolymer molecule in each preparation. It is to beunderstood that the blocks of propylene oxide and ethylene oxide do nothave to be pure. Small amounts of other materials can be admixed so longas the overall physical chemical properties are not substantiallychanged. A more detailed discussion of the preparation of these productsis found in U.S. Pat. No. 2,674,619 which is incorporated herein byreference.

Illustrative ethylene oxide-propylene oxide condensation products whichmay be employed in the preparation of the fibrinolytic composition ofthe present invention include, but are not limited to, the followingcopolymers:

1. A polyol with an average molecular weight of 4700 containingapproximately 80% by weight ethylene oxide.

2. A polyol with an average molecular weight of 3400 containingapproximately 50% by weight ethylene oxide.

3. A polyol with an average molecular weight of 7700 containingapproximately 70% by weight ethylene oxide.

4. A polyol with an average molecular weight of 14,600 containingapproximately 80% by weight ethylene oxide.

5. A polyol with an average molecular weight of 12,600 containingapproximately 70% by weight ethylene oxide.

6. A polyol with an average molecular weight of 9500 containingapproximately 90% by weight ethylene oxide.

The preferred ethylene oxide-propylene oxide copolymer for use in thefibrinolytic composition of the present invention is a copolymer havingthe following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE I

Addition of the copolymer to a clot dissolving enzyme results in asynergistic effect on the clot dissolving activity of the enzyme asdemonstrated in this Example.

Sterile 1 ml tuberculin syringes are packed with 0.6 ml of 500 to 750micron glass beads (Polyscience, Inc., Warington, Pa.). The tips of thesyringes are plugged with nytex filters and a one-way plastic stopcock.Fresh frozen platelet-poor citrated plasma is spiked with 15 μCi/ml¹²⁵ Ihuman fibrinogen (Sigma Chemical Co., St. Louis, Mo.). The radioactiveplasma is diluted 1:2 with normal saline, and recalcified with calcium(American Dade, Aquada, Puerto Rico) at 1 volume of calcium to 4 volumesdiluted plasma.

Radioactive fibrinogen is bound to the glass bead columns as follows:Equal volumes of the radioactivity labelled recalcified plasma are addedto parallel bead columns and allowed to run through the beads with noexcess plasma above the beads. All procedures and manipulations of the"bead clots" are performed at 37° C. The bead/plasma clots are allowedto incubated for 30 minutes, then washed for 30 minutes with normalsaline. During the last 5 minutes of the wash with saline, the flowrates are monitored and columns whose flow rates are abnormally fast orslow are excluded form the experiment. Normal flow rates average 0.2ml/minute.

The copolymer that is used in this example has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The copolymer is prepared as a stock solution of 1%copolymer by weight in normal saline.

Blood containing t-PA, with or without the copolymer, is passed throughthe columns as follows: 10 ml of heparinized whole blood is drawn freshand mixed with t-PA (10 μg in 1 ml normal saline; single chain; SigmaChemical Co. St. Louis, Mo.). A volume of 5.5 ml of blood is mixed witheither 0.5 ml normal saline or 0.5 ml of copolymer stock solution. Onealiquot of a preparation of whole blood or whole blood diluted 1:3 withnormal saline is run on each column. Three ml of each blood preparationis added to a reservoir connected to each column. Fractions arecollected every minute until all flow ceased. The volume in each tube ismeasured and radioactivity counted in a Tracor Analytic gamma counter(TmAnalytic, Inc., Elk Grove Village, Ill.) Appearance of radioactivityin the collection tubes indicates lysis of the clot.

The data are summarized in Table A and FIG. 1. FIG. 1 shows cumulative¹²⁵ I fibrinogen (counts per minute) released from the columns plottedas a function of time.

                  TABLE A                                                         ______________________________________                                        Demonstration of Synergy between                                              Copolymer and t-PA                                                                             Vol-                                                                  Time    ume     Counts Counts                                                                              Counts                                           (Min-   Reco-   Minute Minute                                                                              Minute                                  Perfusate*                                                                             utes)   vered   (Volume)                                                                             (ml)  (Cumulative)                            ______________________________________                                        Blood, t-PA,                                                                           1       0.3      2031   6770 2031                                    Copolymer                                                                              2       0.25     3042   12168                                                                              5073                                             3       0.3     13051   43503                                                                              18124                                            4       0.2     40190  200950                                                                              58314                                            5       0.25    40260  161040                                                                              98574                                            6       0.25    40009  160036                                                                              138583                                  Blood, t-PA                                                                            1       0.15     885    5900  885                                             2       0.2      1330   6650 2215                                             3       0.2      3681   18405                                                                              5896                                             4       0.3     16333   54443                                                                              22229                                            5       0.4     24932   62330                                                                              47161                                            6       0.45    30545   67878                                                                              77706                                            7       0.6     40365   67275                                                                              118071                                  Blood,   2       0.8      340     425  340                                    Copolymer                                                                              3       0.7      351     501  691                                             4       0.6      270     450  961                                             5       0.6      226     377 1187                                             6       0.5      204     408 1391                                             7       0.4      178     445 1569                                    ______________________________________                                    

A simulated thrombus containing ¹²⁵ I fibrin was prepared as describedin the text. The ability of test preparations to dissolve the fibrin wasdetermined by measuring the rate of elution of radioactivity from thecolumn. The copolymer is not an enzyme and has no reactive groups, so itis unable to lyse cross-linked fibrin, but it does increase thefibrinolytic activity of t-PA in this model which was designed tosimulate the structure and flow conditions of a coronary thrombus.

As can be seen from Table A and FIG. 1, treatment of the radioactiveclot with the surface active copolymer releases little of theradioactivity indicating no lysis of the clot. Administration of t-PA tothe clot causes release of radioactivity indicating lysis of the clot inthe column. However, when the surface active copolymer is added to thesolution, the rate of lysis of the clot in the column is dramaticallyincreased. Thus, the combination of surface active polymer and t-PAlysed the clot in the column at a significantly faster rate than didt-PA alone.

In other experiments, the model is modified by changing the size of thebeads, the concentration of plasma used to make the clot, the dilutionof blood or the concentration of enzyme or copolymer. In severalinstances, columns are produced in which whole blood fails to flow atall while blood with copolymer flows at a rate of about 0.05 ml/minute.t-PA in the blood is unable to dissolve any of the fibrin in such clotsas measured by release of ¹²⁵ I label because there is no flow of bloodthrough the clot. The use of copolymer with the blood and t-PA in suchsituations caused rapid lysis of the clot.

EXAMPLE II

The fibrinolytic composition is tested in an ex vivo rat heart model.The detailed design of the system is described elsewhere. (See Paulson,et al., Basic Res. Cardiol., Vol. 81, pp. 180-187, 1986). This modelmeasures the ability of the isolated heart to recover from a 30 to 90minute ischemic period where the flow of nutrients is reduced to 10percent of normal or completely stopped, then followed by a 10 minuteperiod of reperfusion. Three parameters measured: (1) cardiac output(CO); (2) left ventricular systolic pressure (LVSP); and (3) leftventricular contraction (dp/dt). Assessment of heart recovery and amountof damage are discussed in Paulson, D. J. et al. Basic Res. Cardiol.,Vol. 79, pp. 551-561, 1984.

In this experiment, hearts are perfused with washed whole human bloodwith no heparin added. Flow is completely stopped for 30 minutes,followed by 10 minutes reperfusion with washed whole human blood withoutheparin but with the additive or additives indicated in Table B. Thecopolymer that is used in this example has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The copolymer is prepared as a stock solution of 1%copolymer by weight in normal saline.

The results of the test are as follows. The final concentration of thesurface active copolymer used in this Example is 0.68 mg/ml. Thestreptokinase that is used in this Example is obtained from SigmaChemical Company, St. Louis, Mo. Streptokinase is administered at aconcentration of 100 units/heart.

The results are shown in Table B.

                  TABLE B                                                         ______________________________________                                                       Percent Cardiac Recovery                                                      (Values are mean)                                              Additions        CO       LVSP    dp/dt                                       ______________________________________                                        Whole Blood       5       24      10                                          with copolymer.sup.1                                                                           38       82      65                                          with streptokinase.sup.2                                                                       33       75      60                                          with copolymer and                                                                             58.sup.4 88      78                                          Streptokinase                                                                 With SOD.sup.3    9        5       5                                          with copolymer and SOD                                                                         85.sup.5 92      96                                          with copolymer, SOD                                                                            84.sup.5 93      92                                          and streptokinase                                                             ______________________________________                                         .sup.1 The final concentration of copolymer is approximately 0.6 mg/ml.       .sup.2 The amount of streptokinase is approximately 100 units/heart.          .sup.3 The amount of SOD is 3000 units/heart.                                 .sup.4 p < 0.5 for cardiac output (CO) differences between the group of       the combination of (a) Copolymer and Streptokinase and (b) copolymer,         streptokinase and SOD and the group of (a) whole blood ischemic control,      (b) Copolymer only, and (c) Streptokinase only. Student's test was used t     determine differences between independent means. A result of p < 0.5 was      regarded as significant.                                                      .sup.5 Not done                                                          

As can be seen from Table B, the copolymer and streptokinase combinationprotected the heart better than the copolymer or treptokinase alone. Thecombination of SOD and the surface active copolymer protected the heartmuscle from the effects of blood deprivation better than the copolymeralone, SOD or the copolymer streptokinase combination when using cardiacoutput as a measure of heart muscle viability. In addition, coronaryartery resistance showed improvement with the surface active copolymerpresent in the perfusion medium.

EXAMPLE III

For testing a patient weighing about 180 lbs with a pulmonary embolism,reconstitute 500 mg of urakinase (Abbokinase, Abbot Laboratories, NorthChicago, IL.) in 105 ml of sterile water. To the urikinase solution add90 ml of an 0.9% sodium chloride solution containing 6 grams of anethylene oxide-propylene oxide copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound arethoroughly mixed to form a homogeneous solution. The final volume of thesolution is 195 ml.

Administer the fibrinolytic composition of the present invention bymeans of a constant infusion pump that is capable of delivering a totalvolume of 195 ml. A priming dose of the fibrinolytic composition of thepresent invention is administered at a rate of 90 ml/hour over a periodof 10 minutes. This is followed by a continuous infusion of the presentinvention at a rate of 15 ml- hour for 12 hours. Since some of thefibrinolytic composition of the present invention will remain in thetubing at the end of an infusion pump delivery cycle, the remainingsolution is flushed out of the tube by administering a solution of 0.9%sodium chloride at a rate of 15 ml/hour.

EXAMPLE IV

For treating a patient with a coronary artery thrombi, reconstitute 75mg of urokinase (Abbokinase, Abbot Laboratories, North Chicago IL.) in15.6 ml of sterile water. To the urokinase solution add 300 ml of 5%dextrose solution containing 15 grams of an ethylene oxide-propyleneoxide copolymer with the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The urokinase and the copolymer are thoroughly mixedto form a homogeneous solution. The solution is then diluted with 5%dextrose to a final volume of 500 ml.

The solution comprising the present invention is infused into theoccluded artery at a rate of 4 ml per minute for periods up to 2 hours.To determine response to the solution of the fibrinolytic composition ofthe present invention, periodic angiography is performed.

EXAMPLE V

For treating a patient weighing about 180 lbs with a pulmonary embolism,reconstitute 500 mg of urokinase (Abbokinase, Abbot Laboratories, NorthChicago, IL.) in 105 ml of sterile water. To the urokinase solution add90 ml of an 0.9% sodium chloride solution containing 6.0 grams of anethylene oxide-propylene oxide copolymer with the following generalformula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The urokinase and the copolymer are thoroughly mixedto form a homogeneous solution. The solution is then diluted with 0.9%sodium chloride to a final volume of 195 ml.

Administer 137 ml of a 5% isotonic dextrose solution with 3% wt/volethylene oxide-propylene oxide copolymer lysed therein to the patientover a 2 minute period. This gives a blood concentration of copolymer ofapproximately 0.6 mg/ml (assuming blood is 8% of body weight).

The fibrinolytic composition of the present invention is thenimmediately administered by means of a constant infusion pump that iscapable of delivering a total volume of 195 ml. A priming dose of thepresent invention is administered at a rate of 90 ml/hour over a periodof 10 minutes. This is followed by a continuous infusion of the presentinvention at a rate of 15 ml/hour for 12 hours. Since some of thepresent invention will remain in the tubing at the end of an infusionpump delivery cycle, the remaining solution is flushed out of the tubeby administering a solution of 0.9% sodium chloride containing 3.0%copolymer at a rate of 15 ml/hour.

After the clot is lysed, a solution of the copolymer is administered byintravenous drip at a rate of about 25 mg/kg body weight/hour tomaintain a blood concentration of the copolymer of approximately 0.6mg/ml. The administration of the copolymer solution is continued forfour days following the administration of the fibrinolytic compositionof the present invention.

EXAMPLE VI

The effect of the surface active copolymer and an anticoagulant intissue following ischemic damage is demonstrated in this example. Thecomposition comprising the surface active copolymer and ananticoagulant, such as heparin, shows synergistic results. Reconstitute1000 units of heparin (Sigma Chemical Company, St. Louis, MO.) in 200 mlof normal (0.9%) sodium chloride solution and add 1.36 g of thecopolymer of the present invention and resuspend washed whole humanblood to formulate the perfusion medium. The copolymer has the followinggeneral formulation:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

Hearts excised from anesthetized Sprague-Dawley rats were perfused for10 minutes with (a) blood and heparin or with (b) blood, heparin andcopolymer following a 90 minute low-flow ischemia. Cardiac output (CO),left ventricular systolic pressure (LVSP) and left ventricularcontraction (dp/dt) were determined and are expressed as percent ofrecovery as compared to normal hearts. Ischemic animals' hearts whichreceived blood with heparin showed poor recovery: 12% CO, 44% LVSP and34% dp/dt. Hearts given blood, heparin and copolymer showed excellentrecovery: 90% CO, 92% LVSP, and 84% dp/dt. For the heparin withCopolymer group, all three parameters were statistically different(p<0.01) as compared to the ischemic control group (heparin only).Differences between independent means were determined by the Student'stest. This example also illustrates the ability of the copolymer toimprove flow through damaged tissue by virtue of its lubricatingproperties under conditions where there is not thrombus or embolusformation.

EXAMPLE VII

A test is performed to demonstrate the ability of the combination ofsuperoxide dismutase (SOD) and an appropriate copolymer to producegreater protection of ischemic myocardium from reperfusion injuryassociated with oxygen radicals and other factors than SOD alone.

Under general anesthesia (sodium thiopental 25 mg/kg), the dogs areintubated and ventilated with 70% oxygen at a rate of 12 breaths perminute. A satisfactory level of anesthesia is maintained withintermittent boluses of pentothal as required. After skin preparation, aleft anterior thoracotomy is performed, the pericardium incised and theheart exposed. The left anterior descending (LAD) coronary artery isidentified, isolated and encircled with a snare 1 cm from its origin.Temporary LAD occlusion is accomplished by tightening the snare andcontinues for 90 minutes. During the procedure, the heart rate and bloodpressure are monitored utilizing a Hewlett-Packard 7758B 8-channelrecorder. Arterial blood pressure is monitored through an 18 gaugeindwelling catheter in the right femoral artery and measured with aHewlett-Packard quartz transducer. Electrocardiographic evidence foranteroseptal myocardial ischemia is also monitored. Reperfusion of theligated vessel after 90 minutes of ischemia is achieved by a gradualrelease of the snare to prevent the hyperemic response. A defibrillatoris available in the room as are all necessary cardiotonic drugs in theevent of cardiac fibrillation or circulatory collapse due to the LADligation.

Therapeutic agents are infused in conjunction with reperfusion asfollows: bovine superoxide dismutase with approximately 3000 units ofactivity per milligram assayed by the method of McCord, J. Biol. Chem.,Vol. 244, p. 6049 (1969) is obtained from Sigma Chemical Company, St.Louis, MO. It is dissolved in 100 ml of normal saline and infusedintravenously over 90 minutes starting 15 minutes before restoration ofperfusion. This stimulates the effect which occur during lysis of acoronary thrombus. A solution of copolymer is prepared at 2%weight/volume in saline. It is given intravenously as a bolus over 2minutes in a dose sufficient to achieve a blood level of 0.6 mg/mlfollowed by a constant infusion of approximately 25 mg/kg/hour in orderto maintain the blood level of approximately 0.6 mg/ml for the remainderof the experiment.

The ethylene oxide-propylene oxide surface active copolymer has thefollowing general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

The synergistic effect of the combination is demonstrated by comparingthe results of dogs treated with both the copolymer and SOD with thosetreated with either material alone or no treatment.

Agents are infused intravenously utilizing an IVAC 560 infusion pump.Infusion begins 15 minutes prior to release of the snare and continuesuntil the total dose for each group has been administered. The chest isclosed in layers. A chest tube is utilized to evacuate the pneumothoraxand is removed when spontaneous respirations resume. I.V. fluids aregiven (Lactated Ringer's Solution) to compensate for the 24 hour NPOperiod preceding the operation, in addition to a 3 to 1 ratio tocompensate for blood loss. The animals are then maintained and followedclosely for the next 24 hours. Each animal is then returned to theoperating suite and under general anesthesia the previous incision isreopened. The animal is sacrificed utilizing a barbiturate overdose. Theheart and proximal 4 of ascending aorta is excised being sure to includethe origins of the coronary arteriesl.

All groups are subjected to the same procedures for identification ofthe area of the myocardium at risk for infarction and the area actuallyinfarcted.

This technique involves perfusion of the LAD with 2, 3,5-triphenyltetrazolium chloride (TTC), which stains the intactmyocardium red and leaves the infarcted myocardium unstained, The limitsof the area of myocardium at risk are determined by perfusing theremainder of the coronary system, via the aortic root, with Evans Bluedye. The area at risk is defined by a lack of Evans Blue stain.

The combination of the surface active copolymer and superoxide dismutaseis synergistic in protecting myocardial tissue. The amount of tissuedamaged after the ischemic period was significantly less than withsurface active copolymer or mannitol alone.

EXAMPLE VIII

A test is performed to demonstrate the ability of the combination ofmannitol and an appropriate surface active copolymer to produce greaterprotection of ischemic myocardium from reperfusion injury associatedwith oxygen radicals and other factors than mannitol alone.

Under general anesthesia (sodium thiopental 25 mg/kg), the dogs areintubated and ventilated with 70% oxygen at a rate of 12 breaths perminute. A satisfactory level of anesthesia is maintained withintermittent boluses of pentothal as required. After skin preparation, aleft anterior thoracotomy is performed, the pericardium incised and theheart exposed. The left anterior descending (LAD) coronary artery isidentified, isolated and encircled with a snare 1 cm from its origin.Temporary LAD occlusion is accomplished by tightening the snare andcontinues for 90 minutes. During the procedure, the heart rate and bloodpressure are monitored utilizing a Hewlett-Packard 7758B 8-channelrecorder. Arterial blood pressure is monitored through an 18 gaugeindwelling catheter in the right femoral artery and measured with aHewlett-Packard quartz transducer. Electrocardiographic evidence foranteroseptal myocardial ischemia is also monitored. Reperfusion of theligated vessel after 90 minutes of ischemia is achieved by a gradualrelease of the snare to prevent the hyperemic response. A defibrillatoris available in the room as are all necessary cardiotonic drugs in theevent of cardiac fibrillation or circulatory collapse due to the LADligation.

Therapeutic agents are infused in conjunction with reperfusion asfollows: Two ml/kg of a mannitol solution (12.5 g/50 ml of isotonicsaline) (Sigma Chemical Co., St. Louis, MO.) is infused intravenouslyover 45 minutes starting 15 minutes before restoration of perfusion.This stimulates the effects which occur during lysis of a coronarythrombus. A solution of copolymer is prepared at 2% weight/volume insaline. It is given intravenously as a bolus over 2 minutes in a dosesufficient to achieve a blood level of 0.6 mg/ml followed by a constantinfusion of approximately 25 mg/kg/hour in order to maintain the bloodlevel of approximately 0.6 mg/ml for the remainder of the experiment.

The ethylene oxide-propylene oxide surface active copolymer has thefollowing general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

The synergistic effect of the combination is demonstrated by comparingthe results of dogs treated with both the copolymer and mannitol withthose treated with either material alone or no treatment.

Agent are infused intravenously utilizing an IVAC 560 infusion pump.Infusion begins 15 minutes prior to release of the snare and continuesuntil the total dose for each group has been administered. The chest isclosed in layers. A chest tube is utilized to evacuate the pneumothoraxand is removed when spontaneous respirations resume. I.V. fluids aregiven (Lactated Ringer's Solution) to compensate for the 24 hour NPOperiod preceding the operation, in addition to a 3 to 1 ratio tocompensate for blood loss. The animals are then maintained and followedclosely for the next 24 hours. Each animal is then returned to theoperating suite and under general anesthesia the previous incision isreopened. The animal is sacrificed utilizing a barbiturate overdose. Theheart and proximal 4 cm of ascending aorta is excised being sure toinclude the origins of the coronary arteries.

All groups are subjected to the same procedures for identification ofthe area of the myocardium at risk for infarction and the area actuallyinfarcted.

This technique involves perfusion of the LAD with 2, 3,5-triphenyltetrazolium chloride (TTC), which stains the intactmyocardium red and leaves the infarcted myocardium unstained. The limitsof the area of myocardium at risk are determined by perfusing theremainder of the coronary system, via the aortic root, with Evans Bluedye. The area at risk is defined by a lack of Evans Blue stain.

The combination of the surface active copolymer and mannitol issynergistic in protecting myocardial tissue. The amount of tissuedamaged after the ischemic period was significantly less than withsurface active copolymer or mannitol alone.

EXAMPLE IX

Glass beads (500-750 microns in diameter) are packed into tuberculinsyringes and coated with fibrinogen by allowing recalcified citratedhuman plasma to coagulate and cross-link for 60 minutes at 37° C.Heparinized human blood, diluted 1:3 with normal saline with or without0.1% surface active copolymer is then added to the reservoir and allowedto pass through the column by gravity at a pressure of 5 cm of water.The volume of blood flowing through the column over 20 minutes ismeasured.

The surface active copolymer used in this example has the followingformula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

The results of this Example are summarized in FIG. 2. As can be seen inFIG. 2, only approximately 0.2 to 0.3 mls of blood flowed through the invitro clot in twenty minutes. However, when the surface active copolymerwas added to the blood, the flow of blood through the clot was increasedto approximately 4 mls.

EXAMPLE X

The surface active copolymer has a cytoprotective effect. This is shownin the following example. Isolated rat hearts are perfused with packedhuman red blood cells suspended in Krebs-Henseleit buffer at ahematocrit of 12%.

The surface active copolymer used in this example has the followingformula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

After a period of stabilization, the lines are clamped to produce noflow ischemia for one-half hour. The lines are then reopened tofacilitate reperfusion for 10 minutes after which time functionalmeasurements are made and the hearts fixed for histologic examination.

Control hearts reperfused with blood alone recover only 5% of normalfunction as measured by cardiac output. Histologically, they showwidespread contraction bands indicative of myocardial necrosis. Inaddition, there is extensive sloughing of arteriolar endothelial cells.Hearts treated identically except that the surface active copolymer isadded to the blood for the last 10 minutes of the experiment duringreperfusion regain 40% of normal function, show much less evidence ofcontraction band necrosis and preservation of endothelial cells. Under abroad range of experimental conditions, the surface active copolymer canprotect myocardial cells from necrosis associated with reperfusionfollowing a degree of ischemic injury which cannot be tolerated bycontrol hearts. The protective effect of the surface active copolymer isgreatest when it is added early in the experiment and when conditions oflow flow rather than no flow ischemia are used.

EXAMPLE X

Blood was collected using the two-syringe method from healthy volunteerswith normal platelet counts and diluted 1:10 with 0.11M sodium citrate.Either the surface active copolymer or normal saline was then gentlymixed with the blood. The surface active copolymer was used in finalconcentration of 0.6 mg/ml and 2.0 mg/ml. Platelet-rich plasma wasseparated by centrifugation at 200×G for twelve minutes. Platelet-poorplasma was separated by centrifugation at 600×G for twenty minutes. Theplatelet-rich plasma was stored in polypropylene tubes and continuouslyagitated for specified periods of time.

The surface active copolymer used in Examples XII through XVI has thefollowing formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

EXAMPLE XII

In vitro testing of aggregation utilizes an optical aggregometer(Bio-Data Model PAP-2A) and is based on the turbidometric method ofBorn. Platelet-rich plasma (0.45 ml) is warmed to 37° C. while beingagitated continuously with a small magnetic stir bar. Platelet-poorplasma (0.50 ml) is used to blank the platelet-rich plasma. Followingthe addition of an aggregating reagent (0.05 ml), platelets clumptogether, causing the turbidity of the platelet-rich plasma to decrease.The turbidity of the suspension is constantly measured by recordingtransmission of a light beam directed through it, and is recorded as achange in voltage on a chart recorder. The rate of aggregation isdependent on the number of platelets, temperature, concentration ofaggregating reagents, calcium, and fibrinogen. Aggregating agents wereused at the following standard concentrations unless otherwiseindicated: ADP (Dade Cluster Reagent) 2×10⁻⁵ M,; Collagen (Dade ClusterReagent) 200 μg/ml; and, Ristocetin (Bio-Data Corp.) 1.2 mg/ml.

FIG. 3 illustrates the ability of the surface active copolymer, at both0.6 mg/ml and 2.0 mg/ml, to preserve platelet function when using ADP at2×10⁻⁵ M as the agonist. While the percent maximum aggregation isdecreased from the immediate analysis, the surface active copolymerplatelets retained a substantial amount of activity. The saline controldemonstrated either no or very little activity at the 24 and 72 hourassays.

EXAMPLE XIII

FIG. 4 illustrates the preservation effect of the surface activecopolymer over 24 and 72 hours when using collagen as an agonist. Whiledecreased, platelets stored in the presence of the surface activecopolymer still maintains substantial functional activity. The controlplatelets demonstrate no activity after this prolonged storage.

EXAMPLE XIV

Table C shows the results of several experiments which test thefunctional ability of platelets at time of collection and, also, at 24hours storage. The agonist used in this study is ADP at 2×10⁻⁵ M. At 24hours storage, the surface active copolymer at 0.6 mg/ml and 2.0 mg/mlconsistently improves the function of the platelets when compared to thesaline control.

                  TABLE C                                                         ______________________________________                                                   % Maximun aggregation ± SE                                                 Immediate   24 hours                                               ______________________________________                                        Saline control                                                                             89.5 ± 2.4 (n = 4)                                                                       16.5 ± 4.3 (n = 4)                              RheothRx 0.6 mg/ml                                                                         82.7 ± 2.9 (n = 3)                                                                       51.8 ± 6.2 (n = 4)                              RheothRx 0.2 mg/ml                                                                         84.0 ± 1.9 (n = 3)                                                                       57.0 ± 5.6 (n = 4)                              ______________________________________                                    

EXAMPLE XV

Platelet counts on the platelet-rich plasma samples are conducted in theHematology Laboratory at Emory University Hospital. The mean plateletvolume (MPV) and platelet histograms are also analyzed. This laboratoryemploys a Coulter Stacker (Coulter Electronics, Hialeah, FL.) in sampleanalysis. The platelet counts and MPVs remains unchanged in the samplesincubated with the surface active copolymer, as shown in Table D below.The control sample lost a substantial number of platelets over the 24hours. The Coulter Stacker is unable to determine a value for the MPV inthe control sample. However, the platelet histogram shows a definiteshift to the left, indicating a smaller value for the MPV. The platelethistograms for the surface active copolymer samples are unchanged overthis period of time.

                  TABLE D                                                         ______________________________________                                                        Platelets/cumm                                                                          MPV                                                 ______________________________________                                        Immediate         385,000     8.3                                             Saline control - 24 hrs.                                                                        185,000     **                                              0.6 mg/ml RheothRx - 24 hrs.                                                                    408,000     7.8                                             2.0 mg/ml RheothRx - 24 hrs.                                                                    414,000     8.1                                             ______________________________________                                    

EXAMPLE XVI

Two 15 ml polypropylene centrifuge tubes (Becton Dickinson, LincolnPark, N.J.) are filled with approximately 5 ml of whole blood drawn froma healthy human volunteer. To one of the tubes is added the surfaceactive copolymer with the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400. The final concentration of the surface active copolymer in theblood is approximately 0.67 mg/ml. The tubes are slowly inverted severaltimes over a 5 minute period to be sure that the blood in both tubes isin contact with the sides of the tubes. The tubes are then allowed tostand upright. The blood in the tube without the surface activecopolymer uniformly coated the sides of the tube. The tube with thesurface active copolymer did not coat the sides of the tube.

It is believed that the blood in the tube without the surface activecopolymer coats the sides of the tube because of the adhesion ofhydrophobic proteins, such as fibrinogen, to the hydrophobic sides ofthe tube. However, the blood with the surface active copolymer does notcoat the sides of the tube. This is probably because the surface activecopolymer blocks the hydrophobic interaction between the hydrophobicproteins present in the blood and the hydrophobic sides of the tube.

EXAMPLE XVII

The surface active copolymer is effective in promoting blood flow intissue damaged by heat wherein there are increased pathologichydrophobic surfaces.

The ethylene oxide-propylene oxide surface active copolymer used in thisexample has the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

In this example, the backs of 300 g rats are shaved. The rats areanesthetized and then placed in a plastic frame wherein 36 squarecentimeter portion of the shaved back is exposed. The exposed tissue isthen scalded with boiling water for 10 sec. This treatment causes a fullthickness burn on the back. Animals are dryed with a clean towel andplaced into individual cages. Within 30 minutes following the burninjury, control rats receive either 10 ml Ip saline or 1 ml IV isotonicsaline. Rats that receive the surface active copolymer receive either 10ml IP surface active copolymer (200 mg/kg in isotonic saline) or 1 ml IVsurface active copolymer (50 mg/kg in isotonic saline). After 24 hours,the control rats develop large blisters and have a blanched appearance.The copolymer treated rats show no blisters and the gross abnormalitiesdemonstrated in the control rats are significantly less. Thisobservation is made in both the IV and IP treated rats.

The rats are biopsied 24 hours after the burn. The control rats showsevere necrosis in the burn area. The copolymer treated rats show onlypartial thickness damage and show viable cells in the basal layer ofhair follicles. In addition, there is significantly greater blood flowand less loss of fluid in the burned tissue from the copolymer treatedrats.

Although not wanting to be bound by the following theory, it is thoughtthat reperfusion injury mediated by granulocyte adhesion-endothelialcell interaction which causes tissue damage was inhibited by blockingpathological hydrophobic interactions thereby preserving endothelialcells, preventing sludging of blood and preventing loss of fluid intotissue and/or other factors.

EXAMPLE XVIII

The ethylene oxide-propylene oxide surface active copolymer used in thisexample has the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

The surface active copolymer of the present invention has little effecton viscosity of normal blood. However, the surface active copolymer ofthe present invention does have an effect on blood from a patientundergoing trauma such as an operation. This is shown in the followingexperiment. Blood from patients about to undergo open heart surgery iscollected and blood viscosity at various shear rates is measured bothwith and without the surface active copolymer present. The surfaceactive copolymer is present in the blood at a concentration of about 0.6mg/ml.

FIG. 5 shows the viscosity of the blood from preoperative patients withand without the surface active copolymer. The surface active copolymerhas little effect on the viscosity of the blood at all shear ratestested when compared to blood without the copolymer. FIG. 6 shows theviscosity of blood from patients six hours after open heart surgery. Asshown, blood from these patients with no copolymer showed a largeincrease in viscosity at low shear rates. When the copolymer is added tothe blood at a concentration of about 0.6 mg/ml, the viscosity at lowshear rates is reduced to normal levels.

There was a significant increase in soluble fibrin levels inpostoperative patients when compared to preoperative patients. Althoughnot wanting to be bound by the following hypothesis, it is believed thatthe reduction is viscosity of blood at low shear rates is a result ofthe copolymer's ability to reduce pathologic hydrophobic interactions inthe blood with the high concentration of soluble fibrin. This allowsblood to flow more efficiently through the microvasculature.

It should be understood, of course, that the foregoing relates only to apreferred embodiment of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

I claim:
 1. A method for preserving a suspension of platelets comprisingadmixing with the suspension of platelets an effective amount of asurface active copolymer with the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, and b is aninteger such that the hydrophile portion represented by (C₂ H₄ O)constitutes approximately 50% to 95 % by weight of the compound.
 2. Themethod of claim 1 wherein said surface active copolymer has thefollowing formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 1200 to 3500, and b is aninteger such that the hydrophile portion represented by (C₂ H₄ O)constitutes approximately 50% to 95% by weight of the compound.
 3. Themethod of claim 1 wherein said surface active copolymer comprises thefollowing formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400.